Antisense oligonucleotide modulation of raf gene expression

ABSTRACT

Oligonucleotides are provided which are targeted to nucleic acids encoding human c-raf and capable of inhibiting raf expression. The oligonucleotides may have chemical modifications at one or more positions and may be chimeric oligonucleotides. Methods of inhibiting the expression of human raf using oligonucleotides of the invention are also provided. The present invention further comprises methods of inhibiting hyperproliferation of cells and methods of treating abnormal proliferative conditions which employ oligonucleotides of the invention.

This application is a continuation-in-part of Ser. No. 08/250,856, filedMay 31, 1994, now U.S. Pat. No. 5,563,255.

FIELD OF THE INVENTION

This invention relates to compositions and methods for modulatingexpression of the raf gene, a naturally present cellular gene which hasbeen implicated in abnormal cell proliferation and tumor formation. Thisinvention is also directed to methods for inhibiting hyperproliferationof cells; these methods can be used diagnostically or therapeutically.Furthermore, this invention is directed to treatment of conditionsassociated with expression of the raf gene.

BACKGROUND OF THE INVENTION

Alterations in the cellular genes which directly or indirectly controlcell growth and differentiation are considered to be the main cause ofcancer. The raf gene family includes three highly conserved genes termedA-, B- and c-raf (also called raf-1). Raf genes encode protein kinasesthat are thought to play important regulatory roles in signaltransduction processes that regulate cell proliferation. Expression ofthe c-raf protein is believed to play a role in abnormal cellproliferation since it has been reported that 60% of all lung carcinomacell lines express unusually high levels of c-raf mRNA and protein. Rappet al., The Oncogene Handbook, E. P. Reddy, A. M Skalka and T. Curran,eds., Elsevier Science Publishers, New York, 1988, pp. 213-253.

Oligonucleotides have been employed as therapeutic moieties in thetreatment of disease states in animals and man. For example, workers inthe field have now identified antisense, triplex and otheroligonucleotide compositions which are capable of modulating expressionof genes implicated in vital, fungal and metabolic diseases.

As examples, U.S. Pat. No. 5,135,917, issued August 4, 1992, providesantisense oligonucleotides that inhibit human interleukin-1 receptorexpression. U.S. Pat. No. 5,098,890, issued Mar. 24, 1992, in the nameof Gewirtz et al., is directed to antisense oligonucleotidescomplementary to the c-myb oncogene and antisense oligonucleotidetherapies for certain cancerous conditions. U.S. Pat. No. 5,087,617,issued Feb. 11, 1992, provides methods for treating cancer patients withantisense oligonucleotides. U.S. Pat. No. 5,166,195 issued Nov. 24,1992, provides oligonucleotide inhibitors of HIV. U.S. Pat. No.5,004,810, issued Apr. 2, 1991, provides oligomers capable ofhybridizing to herpes simplex virus Vmw65 mRNA and inhibitingreplication. U.S. Pat. No. 5,194,428, issued Mar. 16, 1993, providesantisense oligonucleotides having antiviral activity againstinfluenzavirus. U.S. Pat. No. 4,806,463, issued February 21, 1989,provides antisense oligonucleotides and methods using them to inhibitHTLV-III replication. U.S. Pat. No. 5,286,717 (Cohen et al.), issuedFeb. 15, 1994, is directed to a mixed linkage oligonucleotidephosphorothioates complementary to an oncogene; U.S. Pat. No. 5,276,019and U.S. Pat. No. 5,264,423 (Cohen et al.) are directed tophosphorothioate oligonucleotide analogs used to prevent replication offoreign nucleic acids in cells. Antisense oligonucleotides have beensafely administered to humans and clinical trials of several antisenseoligonucleotide drugs, targeted both to vital and cellular geneproducts, are presently underway. The phosphorothioate oligonucleotide,ISIS 2922, has been shown to be effective against cytomegalovirusretinitis in AIDS patients. BioWorld Today, Apr. 29, 1994, p. 3. It isthus established that oligonucleotides can be useful therapeuticinstrumentalities and can be configured to be useful in treatmentregimes for treatment of cells and animal subjects, especially humans.

Antisense oligonucleotide inhibition of gene expression has proven to bea useful tool in understanding the roles of raf genes. An antisenseoligonucleotide complementary to the first six codons of human c-raf hasbeen used to demonstrate that the mitogenic response of T cells tointerleukin-2 (IL-2) requires c-raf. Cells treated with theoligonucleotide showed a near-total loss of c-raf protein and asubstantial reduction in proliferative response to IL-2. Riedel et al.,Eur. J. Immunol. 1993, 23, 3146-3150. Rapp et al. have disclosedexpression vectors containing a raf gene in an antisense orientationdownstream of a promoter, and methods of inhibiting raf expression byexpressing an antisense Raf gene or a mutated Raf gene in a cell. WOapplication 93/04170. An antisense oligodeoxyribonucleotidecomplementary to codons 1-6 of murine c-Raf has been used to abolishinsulin stimulation of DNA synthesis in the rat hepatoma cell lineH4IIE. Tornkvist et al., J. Biol. Chem. 1994, 269, 13919-13921. WOApplication 93/06248 discloses methods for identifying an individual atincreased risk of developing cancer and for determining a prognosis andproper treatment of patients afflicted with cancer comprising amplifyinga region of the c-raf gene and analyzing it for evidence of mutation.

Denner et al. disclose antisense polynucleotides hybridizing to the genefor raf, and processes using them. WO 94/15645. Oligonucleotideshybridizing to human and rat raf sequences are disclosed.

Iversen et al. disclose heterotypic antisense oligonucleotidescomplementary to raf which are able to kill ras-activated cancer cells,and methods of killing raf-activated cancer cells. Numerousoligonucleotide sequences are disclosed, none of which are actuallyantisense oligonucleotide sequences.

There remains a long-felt need for improved compositions and methods forinhibiting raf gene expression.

SUMMARY OF THE INVENTION

The present invention provides oligonucleotides which are targeted tonucleic acids encoding human raf and are capable of inhibiting rafexpression. The present invention also provides chimericoligonucleotides targeted to nucleic acids encoding human raf. Theoligonucleotides of the invention are believed to be useful bothdiagnostically and therapeutically, and are believed to be particularlyuseful in the methods of the present invention.

The present invention also comprises methods of inhibiting theexpression of human raf, particularly the abnormal expression of raf.These methods are believed to be useful both therapeutically anddiagnostically as a consequence of the association between rafexpression and hyperproliferation. These methods are also useful astools, for example for detecting and determining the role of rafexpression in various cell functions and physiological processes andconditions and for diagnosing conditions associated with raf expression.

The present invention also comprises methods of inhibitinghyperproliferation of cells using oligonucleotides of the invention.These methods are believed to be useful, for example in diagnosingraf-associated cell hyperproliferation. Methods of treating abnormalproliferative conditions are also provided. These methods employ theoligonucleotides of the invention. These methods are believed to beuseful both therapeutically and as clinical research and diagnostictools.

DESCRIPTION OF THE DRAWING

FIGS. 1A and 1B are line graphs showing the effect of ISIS 5132 (FIG.1A) and a scrambled control oligonucleotide ISIS 10353 (FIG. 1B) ongrowth of A549 lung tumor xenografts in nude mice. ISIS 5132 decreasedtumor size at all doses (0.006 mg/kg; 0.06 mg/kg; 0.6 mg/kg; and 6.0mg/kg) in a dose-dependent manner. The scrambled raf oligonucleotide,ISIS 10353, had no effect at any dose (FIG. 1B).

DETAILED DESCRIPTION OF THE INVENTION

Malignant tumors develop through a series of stepwise, progressivechanges that lead to the loss of growth control characteristic of cancercells, i.e., continuous unregulated proliferation, the ability to invadesurrounding tissues, and the ability to metastasize to different organsites. Carefully controlled in vitro studies have helped define thefactors that characterize the growth of normal and neoplastic cells andhave led to the identification of specific proteins that control cellgrowth and differentiation. The raf genes are members of a gene familywhich encode related proteins termed A-, B- and c-raf. Raf genes codefor highly conserved serine-threonine-specific protein kinases. Theseenzymes are differentially expressed; c-raf, the most thoroughlycharacterized, is expressed in all organs and in all cell lines thathave been examined. A- and B-raf are expressed in urogenital and braintissues, respectively. c-raf protein kinase activity and subcellulardistribution are regulated by mitogens via phosphorylation. Variousgrowth factors, including epidermal growth factor, acidic fibroblastgrowth factor, platelet-derived growth factor, insulin,granulocyte-macrophage colony-stimulating factor, interleukin-2,interleukin-3 and erythropoietin, have been shown to inducephosphorylation of c-raf. Thus, c-raf is believed to play a fundamentalrole in the normal cellular signal transduction pathway, coupling amultitude of growth factors to their net effect, cellular proliferation.

Certain abnormal proliferative conditions are believed to be associatedwith raf expression and are, therefore, believed to be responsive toinhibition of raf expression. Abnormally high levels of expression ofthe raf protein are also implicated in transformation and abnormal cellproliferation. These abnormal proliferative conditions are also believedto be responsive to inhibition of raf expression. Examples of abnormalproliferative conditions are hyperproliferative disorders such ascancers, tumors, hyperplasias, pulmonary fibrosis, anglogenesis,psoriasis, atherosclerosis and smooth muscle cell proliferation in theblood vessels, such as stenosis or restenosis following angioplasty. Thecellular signalling pathway of which raf is a part has also beenimplicated in inflammatory disorders characterized by T-cellproliferation (T-cell activation and growth), such as tissue graftrejection, endotoxin shock, and glomerular nephritis, for example.

It has now been found that elimination or reduction of raf geneexpression may halt or reverse abnormal cell proliferation. This hasbeen found even in when levels of raf expression are not abnormallyhigh. There is a great desire to provide compositions of matter whichcan modulate the expression of the raf gene. It is greatly desired toprovide methods of detection of the raf gene in cells, tissues andanimals. It is also desired to provide methods of diagnosis andtreatment of abnormal proliferative conditions associated with abnormalraf gene expression. In addition, kits and reagents for detection andstudy of the raf gene are desired. "Abnormal" raf gene expression isdefined herein as abnormally high levels of expression of the rafprotein, or any level of raf expression in an abnormal proliferativecondition or state.

The present invention employs oligonucleotides targeted to nucleic acidsencoding raf. This relationship between an oligonucleotide and itscomplementary nucleic acid target to which it hybridizes is commonlyreferred to as "antisense". "Targeting" an oligonucleotide to a chosennucleic acid target, in the context of this invention, is a multistepprocess. The process usually begins with identifying a nucleic acidsequence whose function is to be modulated. This may be, as examples, acellular gene (or mRNA made from the gene) whose expression isassociated with a particular disease state, or a foreign nucleic acidfrom an infectious agent. In the present invention, the target is anucleic acid encoding raf; in other words, the raf gene or mRNAexpressed from the raf gene. The targeting process also includesdetermination of a site or sites within the nucleic acid sequence forthe oligonucleotide interaction to occur such that the desired effect-modulation of gene expression- will result. Once the target site orsites have been identified, oligonucleotides are chosen which aresufficiently complementary to the target, i.e., hybridize sufficientlywell and with sufficient specificity, to give the desired modulation.

In the context of this invention "modulation" means either inhibition orstimulation. Inhibition of raf gene expression is presently thepreferred form of modulation. This modulation can be measured in wayswhich are routine in the art, for example by Northern blot assay of mRNAexpression or Western blot assay of protein expression as taught in theexamples of the instant application. Effects on cell proliferation ortumor cell growth can also be measured, as taught in the examples of theinstant application. "Hybridization", in the context of this invention,means hydrogen bonding, also known as Watson-Crick base pairing, betweencomplementary bases, usually on opposite nucleic acid strands or tworegions of a nucleic acid strand. Guanine and cytosine are examples ofcomplementary bases which are known to form three hydrogen bonds betweenthem. Adenine and thymine are examples of complementary bases which formtwo hydrogen bonds between them. "Specifically hybridizable" and"complementary" are terms which are used to indicate a sufficient degreeof complementarity such that stable and specific binding occurs betweenthe DNA or RNA target and the oligonucleotide. It is understood that anoligonucleotide need not be 100% complementary to its target nucleicacid sequence to be specifically hybridizable. An oligonucleotide isspecifically hybridizable when binding of the oligonucleotide to thetarget interferes with the normal function of the target molecule tocause a loss of utility, and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the oligonucleotide tonon-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment or, in the case of in vitro assays,under conditions in which the assays are conducted.

In preferred embodiments of this invention, oligonucleotides areprovided which are targeted to mRNA encoding c-raf. In accordance withthis invention, persons of ordinary skill in the art will understandthat mRNA includes not only the coding region which carries theinformation to encode a protein using the three letter genetic code, butalso associated ribonucleotides which form a region known to suchpersons as the 5'-untranslated region, the 3'-untranslated region, the5' cap region, intron regions and intron/exon or splice junctionribonucleotides. Thus, oligonucleotides may be formulated which aretargeted wholly or in part to these associated ribonucleotides as wellas to the coding ribonucleotides. The functions of messenger RNA to beinterfered with include all vital functions such as translocation of theRNA to the site for protein translation, actual translation of proteinfrom the RNA, splicing or maturation of the RNA and possibly evenindependent catalytic activity which may be engaged in by the RNA. Theoverall effect of such interference with the RNA function is to causeinterference with raf protein expression.

The present invention provides oligonucleotides for modulation of rafgene expression. Such oligonucleotides are targeted to nucleic acidsencoding c-raf.

In the context of this invention, the term "oligonucleotide" refers toan oligomer or polymer of nucleotide or nucleoside monomers consistingof naturally occurring bases, sugars and intersugar (backbone) linkages.The term "oligonucleotide" also includes oligomers comprisingnon-naturally occurring monomers, or portions thereof, which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of properties such as, for example,enhanced cellular uptake and increased stability in the presence ofnucleases.

The oligonucleotides may be chimeric oligonucleotides. "Chimericoligonucleotides" or "chimeras", in the context of this invention, areoligonucleotides which contain two or more chemically distinct regions,each made up of at least one nucleotide. These oligonucleotidestypically contain at least one region of modified nucleotides thatconfers one or more beneficial properties (such as, for example,increased nuclease resistance, increased uptake into cells, increasedbinding affinity for the RNA target) and a region that is a substratefor RNase H cleavage. In one preferred embodiment, a chimeticoligonucleotide comprises at least one region modified to increasetarget binding affinity, and, usually, a region that acts as a substratefor RNAse H. Affinity of an oligonucleotide for its target (in this casea nucleic acid encoding raf) is routinely determined by measuring the Tmof an oligonucleotide/target pair, which is the temperature at which theoligonucleotide and target dissociate; dissociation is detectedspectrophotometrically. The higher the Tm, the greater the affinity ofthe oligonucleotide for the target. In a more preferred embodiment, theregion of the oligonucleotide which is modified to increase raf mRNAbinding affinity comprises at least one nucleotide modified at the 2'position of the sugar, most preferably a 2'-O-alkyl or2'-fluoro-modified nucleotide. Such modifications are routinelyincorporated into oligonucleotides and these oligonucleotides have beenshown to have a higher Tm (i.e., higher target binding affinity) than2'-deoxyoligonucleotides against a given target. The effect of suchincreased affinity is to greatly enhance antisense oligonucleotideinhibition of raf gene expression. RNAse H is a cellular endonucleasethat cleaves the RNA strand of RNA:DNA duplexes; activation of thisenzyme therefore results in cleavage of the RNA target, and thus cangreatly enhance the efficiency of antisense inhibition. Cleavage of theRNA target can be routinely demonstrated by gel electrophoresis. Inanother preferred embodiment, the chimetic oligonucleotide is alsomodified to enhance nuclease resistance. Cells contain a variety of exo-and endo-nucleases which can degrade nucleic acids. A number ofnucleotide and nucleoside modifications have been shown to make theoligonucleotide into which they are incorporated more resistant tonuclease digestion than the native oligodeoxynucleotide. Nucleaseresistance is routinely measured by incubating oligonucleotides withcellular extracts or isolated nuclease solutions and measuring theextent of intact oligonucleotide remaining over time, usually by gelelectrophoresis. Oligonucleotides which have been modified to enhancetheir nuclease resistance survive intact for a longer time thanunmodified oligonucleotides. A variety of oligonucleotide modificationshave been demonstrated to enhance or confer nuclease resistance.Oligonucleotides which contain at least one phosphorothioatemodification are presently preferred. In some cases, oligonucleotidemodifications which enhance target binding affinity are also,independently, able to enhance nuclease resistance.

Specific examples of some preferred oligonucleotides envisioned for thisinvention may contain phosphorothioates, phosphotriesters, methylphosphonates, short chain alkyl or cycloalkyl intersugar linkages orshort chain heteroatomic or heterocyclic intersugar ("backbone" )linkages. Most preferred are phosphorothioates and those with CH₂--NH--O--CH₂, CH₂ --N(CH₃)--O-- CH₂ (known as the methylene(methylimino) or MMI backbone), CH₂ -O--N(CH₃)--CH₂, CH₂ --N (CH₃)--N(CH₃)--CH₂ and O--N(CH₃)--CH₂ --CH₂ backbones (where phosphodiester isO--P--O--CH₂) . Also preferred are oligonucleotides having morpholinobackbone structures. Summerton, J. E. and Weller, D. D., U.S. Pat. No.5,034,506. In other preferred embodiments, such as the protein-nucleicacid or peptide-nucleic acid (PNA) backbone, the phosphodiester backboneof the oligonucleotide may be replaced with a polyamide backbone, thebases being bound directly or indirectly to the aza nitrogen atoms ofthe polyamide backbone. P. E. Nielsen, M. Egholm, R. H. Berg, O.Buchardt, Science 1991, 254, 1497. Other preferred oligonucleotides maycontain alkyl and halogen-substituted sugar moieties comprising one ofthe following at the 2' position: OH, SH, SCH₃, F, OCN, OCH₃ OCH₃, OCH₃O(CH₂)_(n) CH₃, O(CH₂)_(n) NH₂ or O(CH₂)_(n) CH₃ where n is from 1 toabout 10; C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl oraralkyl; Cl; Br; CN; CF₃ ; OCF₃ ; O--, S--, or N-alkyl; O--, S--, orN-alkenyl; SOCH₃ ; SO₂ CH₃ ; ONO₂ ; NO₂ ; N₃ ; NH₂ ; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a cholesteryl group; a conjugate; a reportergroup; an intercalator; a group for improving the pharmacokineticproperties of an oligonucleotide; or a group for improving thepharmacodynamic properties of an oligonucleotide and other substituentshaving similar properties. Oligonucleotides may also have sugar mimeticssuch as cyclobutyls in place of the pentofuranosyl group. Otherpreferred embodiments may include at least one modified base form or"universal base" such as inosine.

The oligonucleotides in accordance with this invention preferably arefrom about 8 to about 50 nucleotides in length. In the context of thisinvention it is understood that this encompasses non-naturally occurringoligomers as hereinbefore described, having 8 to 50 monomers.

The oligonucleotides used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including Applied Biosystems. Any other means for such synthesismay also be employed; the actual synthesis of the oligonucleotides iswell within the talents of the routineer. It is also well known to usesimilar techniques to prepare other oligonucleotides such as thephosphorothioates and alkylated derivatives. It is also well known touse similar techniques and commercially available modified amidites andcontrolled-pore glass (CPG) products such as biotin, fluorescein,acridine or psoralen-modified amidites and/or CPG (available from GlenResearch, Sterling Va.) to synthesize fluorescently labeled,biotinylated or other modified oligonucleotides such ascholesterol-modified oligonucleotides.

It has now been found that certain oligonucleotides targeted to portionsof the c-raf mRNA are particularly useful for inhibiting raf expressionand for interfering with cell hyperproliferation. Methods for inhibitingc-raf expression using antisense oligonucleotides are, likewise, usefulfor interfering with cell hyperproliferation. In the methods of theinvention, tissues or cells are contacted with oligonucleotides. In thecontext of this invention, to "contact" tissues or cells with anoligonucleotide or oligonucleotides means to add the oligonucleotide(s),usually in a liquid carrier, to a cell suspension or tissue sample,either in vitro or ex vivo, or to administer the oligonucleotide(s) tocells or tissues within an animal.

For therapeutics, methods of inhibiting hyperproliferation of cells andmethods of treating abnormal proliferative conditions are provided. Theformulation of therapeutic compositions and their subsequentadministration is believed to be within the skill in the art. Ingeneral, for therapeutics, a patient suspected of needing such therapyis given an oligonucleotide in accordance with the invention, commonlyin a pharmaceutically acceptable carrier, in amounts and for periodswhich will vary depending upon the nature of the particular disease, itsseverity and the patient's overall condition. The pharmaceuticalcompositions of this invention may be administered in a number of waysdepending upon whether local or systemic treatment is desired, and uponthe area to be treated. Administration may be topical (includingophthalmic, vaginal, rectal, intranasal), oral, or parenteral, forexample by intravenous drip, intravenous injection or subcutaneous,intraperitoneal or intramuscular injection.

Formulations for topical administration may include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives.

In addition to such pharmaceutical carriers, cationic lipids may beincluded in the formulation to facilitate oligonucleotide uptake. Onesuch composition shown to facilitate uptake is Lipofectin (BRL, BethesdaMd.).

Dosing is dependent on severity and responsiveness of the condition tobe treated, with course of treatment lasting from several days toseveral months or until a cure is effected or a diminution of diseasestate is achieved. Optimal dosing schedules can be calculated frommeasurements of drug accumulation in the body. Persons of ordinary skillcan easily determine optimum dosages, dosing methodologies andrepetition rates. Optimum dosages may vary depending on the relativepotency of individual oligonucleotides, and can generally be calculatedbased on EC50's in in vitro and in vivo animal studies. For example,given the molecular weight of compound (derived from oligonucleotidesequence and chemical structure) and an effective dose such as an IC50,for example (derived experimentally), a dose in mg/kg is routinelycalculated.

The present invention is also suitable for diagnosing abnormalproliferative states in tissue or other samples from patients suspectedof having a hyperproliferative disease such as cancer, psoriasis orblood vessel restenosis or atherosclerosis. The ability of theoligonucleotides of the present invention to inhibit cell proliferationmay be employed to diagnose such states. A number of assays may beformulated employing the present invention, which assays will commonlycomprise contacting a tissue sample with an oligonucleotide of theinvention under conditions selected to permit detection and, usually,quantitation of such inhibition. Similarly, the present invention can beused to distinguish raf-associated tumors from tumors having otheretiologies, in order that an efficacious treatment regime can bedesigned.

The oligonucleotides of this invention may also be used for researchpurposes. Thus, the specific hybridization exhibited by theoligonucleotides may be used for assays, purifications, cellular productpreparations and in other methodologies which may be appreciated bypersons of ordinary skill in the art.

The oligonucleotides of the invention are also useful for detection anddiagnosis of raf expression. For example, radiolabeled oligonucleotidescan be prepared by ³² p labeling at the 5' end with polynucleotidekinase. Sambrook et al., Molecular Cloning. A Laboratory Manual, ColdSpring Harbor Laboratory Press, 1989, Volume 2, p. 10.59. Radiolabeledoligonucleotides are then contacted with tissue or cell samplessuspected of raf expression and the sample is washed to remove unboundoligonucleotide. Radioactivity remaining in the sample indicates boundoligonucleotide (which in turn indicates the presence of raf) and can bequantitated using a scintillation counter or other routine means.Radiolabeled oligo can also be used to perform autoradiography oftissues to determine the localization, distribution and quantitation ofraf expression for research, diagnostic or therapeutic purposes. In suchstudies, tissue sections are treated with radiolabeled oligonucleotideand washed as described above, then exposed to photographic emulsionaccording to routine autoradiography procedures. The emulsion, whendeveloped, yields an image of silver grains over the regions expressingraf. Quantitation of the silver grains permits raf expression to bedetected.

Analogous assays for fluorescent detection of raf expression can bedeveloped using oligonucleotides of the invention which are conjugatedwith fluorescein or other fluorescent tag instead of radiolabeling. Suchconjugations are routinely accomplished during solid phase synthesisusing fluorescently labeled amidites or CPG (e.g., fluorescein-labeledamidites and CPG available from Glen Research, Sterling Va. See 1993Catalog of Products for DNA Research, Glen Research, Sterling Va., p.21).

Each of these assay formats is known in the art. One of skill couldeasily adapt these known assays for detection of raf expression inaccordance with the teachings of the invention providing a novel anduseful means to detect raf expression.

Oligonucleotide Inhibition of c-raf Expression

Phosphorothioate deoxyoligonucleotides targeted to human c-raf weredesigned using the Genbank c-raf sequence HUMRAFR (Genbank listingx03484), synthesized and tested for inhibition of c-raf mRNA expressionin T24 bladder carcinoma cells using a Northern blot assay.Oligonucleotide ISIS 5132 (TCQCGCCTGTGACATGCATT; SEQ ID NO: 1), targetedto the 3'UTR of c-raf, showed greater than 90% inhibition and is highlypreferred.

Specificity of ISIS 5132 for raf

Specificity of ISIS 5132 for raf mRNA was demonstrated by a Northernblot assay in which this oligonucleotide was tested for the ability toinhibit Ha-ras mRNA as well as c-raf mRNA in T24 cells. Ha-ras is acellular oncogene which is implicated in transformation andtumorigenesis. ISIS 5132 was shown to abolish c-raf mRNA almostcompletely with no effect on Ha-ras mRNA levels.

Chimeric Oligonucleotides

Chimeric oligonucleotides having SEQ ID NO: 1 were prepared. Theseoligonucleotides had central "gap" regions of 6, 8, or 10deoxynucleotides flanked by two regions of 2'-O-methyl modifiednucleotides. Backbones were uniformly phosphorothioate. In Northern blotanalysis, all three of these oligonucleotides (ISIS 6720, 6-deoxy gap;ISIS 6717, 8-deoxy gap; ISIS 6729, 10-deoxy gap) showed greater than 70%inhibition of c-raf mRNA expression in T24 cells. These oligonucleotidesare preferred. The 8-deoxy gap compound (6717) showed greater than 90%inhibition and is more preferred. Oligonucleotides of SEQ ID NO: 1having an 8-deoxynucleotide gap flanked by either 2'-O-propyl or 2'fluoro modified nucleotides were also active and these compounds arealso preferred.

A chimeric oligonucleotide having SEQ ID NO: 1 with 2'-O-propyl sugarmodifications and chimeric P=O/P=S backbones were also synthesized. Thisoligonucleotide, ISIS 9271, is shown as follows: TCCCGCCTGTGACATGCATTItalic regions indicate regions which are both 2'-modified and havephosphodiester backbones.

Inhibition of Cancer Cell Proliferation

The phosphorothioate oligonucleotide ISIS 5132 (SEQ ID NO: 1) was shownto inhibit T24 bladder cancer cell proliferation. Cells were treatedwith various concentrations of oligonucleotide in conjunction withlipofectin (cationic lipid which increases uptake of oligonucleotide). Adose-dependent inhibition of cell proliferation was demonstrated, asindicated in Table 1, in which "None" indicates untreated control (nooligonucleotide) and "Control" indicates treatment with negative controloligonucleotide. Results are shown as percent inhibition compared tountreated control.

                  TABLE 1                                                         ______________________________________                                        Inhibition of T24 Cell Proliferation by ISIS 5132                             Oligo conc.                                                                              None         Control 5132                                          ______________________________________                                         50 nM     0            +9%     23%                                           100 nM     0            +4%     24%                                           250 nM     0            10%     74%                                           500 nM     0            18%     82%                                           ______________________________________                                    

Effect of ISIS 5132 on T24 Human Bladder Carcinoma T-tumors

Subcutaneous human T24 bladder carcinoma xenografts in nude mice wereestablished and treated with ISIS 5132 and an unrelated controlphosphorothioate oligonucleotide administered intraperitoneally threetimes weekly at a dosage of 25 mg/kg. In this preliminary study, ISIS5132 inhibited tumor growth after eleven days by 35% compared tocontrols. Oligonucleotide-treated tumors remained smaller than controltumors throughout the course of the study.

Effect of ISIS 5132 on MDA-MB 231 Human Breast Carcinoma Tumors

Subcutaneous human MDA-MB 231 breast carcinoma xenografts in nude micewere established and treated with ISIS 5132 and an unrelated controlphosphorothioate oligonucleotide administered intravenously once per dayat a dosage of 0.6 mg/kg or 6.0 mg/kg. ISIS 5132 inhibited tumor growthafter 27 days (end of study) by approximately 80% compared to controls.

ISIS 5132 was also effective when administered intraperitoneally toMDA-MB 231 xenografts in nude mice. Oligonucleotide was administeredonce per day at 0.6 mg/kg or 6.0 mg/kg. By day 27 (end of study), tumorvolume was inhibited by 57% (0.6 mg/kg dose) or 64% (6.0 mg/kg) comparedto control.

Effect of ISIS 5132 on c-raf RNA Levels in MDA-MB231 Tumors

RNA was isolated from MDA-MD231 tumor xenografts and Northern blots wereperformed to evaluate oligonucleotide effects on raf RNA levels. ISIS5132 decreased raf RNA levels after 27 days by 67% when givenintravenously and 39% when given intraperitoneally (both at 6 mg/kg).

Effect of ISIS 5132 on Colo 205 Human Colon Carcinoma Tumors

Subcutaneous human Colo 205 colon carcinoma xenografts in nude mice wereestablished and treated with ISIS 5132 and an unrelated controlphosphorothioate oligonucleotide administered intravenously once per dayat a dosage of 6.0 mg/kg. In this study, ISIS 5132 inhibited tumorgrowth after 25 days by over 40% compared to controls.

Effect of ISIS 5132 on A549 Human Lung Adenocarcinoma Tumors

Subcutaneous human A549 lung adenocarcinoma xenografts were establishedin male Balb/c nude mice and treated with ISIS 5132 and a controloligonucleotide administered daily by intravenous injection at dosesranging from 0.006 to 6.0 mg/kg. ISIS 5132 decreased tumor size at alldoses in a dose-dependent manner, as shown in FIG. 1A. A scrambled rafoligonucleotide, ISIS 10353, had no effect at any dose (FIG. 1B).

Effect of ISIS 5132 on c-raf RNA Levels in A549 Tumor Cells

RNA was isolated from A549 tumor xenografts and Northern blots wereperformed to evaluate oligonucleotide effects on raf RNA levels. ISIS5132 progressively decreased raf RNA levels beginning 8 hours afterstart of oligo treatment. When the experiment was terminated at day 13,RNA levels were still declining and had reached levels approximately 15%of control.

Effect of a 2'-O-methyl Gapped Oligonucleotide with SEQ ID NO: I on A549Lung Xenograft Tumors

ISIS 6717, a 2'-O-methyl gapped oligonucleotide having SEQ ID NO: 1, wascompared to ISIS 5132 (phosphorothioate deoxyoligonucleotide of the samesequence) for ability to inhibit A549 tumor xenograft growth. At dosesof 0.006, 0.06, 0.6 and 6.0 mg/kg given intravenously, the twooligonucleotides were virtually indistinguishable in their effects ontumor growth. ISIS 6717 is therefore a preferred embodiment of thisinvention.

Identification of Oligonucleotides Targeted to Rat and Mouse c-raf

Many conditions which are believed to be mediated by raf kinase are notamenable to study in humans. For example, tissue graft rejection is acondition which is likely to be ameliorated by interference with rafexpression; but, clearly, this must be evaluated in animals rather thanhuman transplant patients. Another such example is restenosis. Theseconditions can be tested in animal models, however, such as the rat andmouse models used here. These data provide added evidence of thetherapeutic utility of antisense oligonucleotides targeted to c-raf.

Oligonucleotide sequences for inhibiting c-raf expression in rat andmouse cells were identified. Rat and mouse c-raf genes have regions ofhigh homology; a series of oligonucleotides which target both rat andmouse c-tar mRNA sequence were designed and synthesized, usinginformation gained from evaluation of oligonucleotides targeted to humanc-raf. These oligonucleotides were screened for activity in mouse bENDcells and rat A-10 cells using Northern blot assays. Twooligonucleotides, ISIS 11061 and ISIS 10707, were found to inhibit c-rafRNA levels by greater than 90% in mouse bEND cells at a dose of 400 nM.These two oligonucleotides inhibited raf RNA levels virtually entirelyin rat A-10 cells at a concentration of 200 nM. The IC50 for ISIS 10707was found to be 170 nM in mouse bEND cells and 85 nM in rat A-10 cells.The IC50 for ISIS 11061 was determined to be 85 nM in mouse bEND cellsand 30 nM in rat A-10 cells.

Effect of ISIS-11061 on Endogenous c-raf mRNA Expression in Mice

Mice were injected intraperitoneally with ISIS 11061 (50 mg/kg) orcontrol oligonucleotide or saline control once daily for three days.Animals were sacrificed and organs were analyzed for c-raf mRNAexpression by Northern blot analysis. ISIS 11061 was found to decreaselevels of c-raf mRNA in liver by approximately 70%. Controloligonucleotides had no effects on c-raf expression. The effect of ISIS11061 was specific for c-raf; A-raf and G3PDH RNA levels were unaffectedby oligonucleotide treatment.

Antisense oligonucleotide to c-raf Increases Survival in Murine HeartAllograft Model

To determine the therapeutic effects of the c-raf antisenseoligonucleotide ISIS 11061 in preventing allograft rejection, thisoligonucleotide was tested for activity in a murine vascularizedheterotopic heart transplant model. Hearts from C57BI10 mice weretransplanted into the abdominal cavity of C3H mice as primaryvascularized grafts essentially as described by Isobe et al.,Circulation 1991, 84, 1246-1255. Oligonucleotides were administered bycontinuous intravenous administration via a 7-day Alzet pump. The meanallograft survival time for untreated mice was 7.83±0.75 days (7, 7, 8,8, 8, 9 days). Allografts in mice treated for 7 days with 20 mg/kg or 40mg/kg ISIS 11061 all survived at least 11 days (11, 11, 12 days for 20mg/kg dose and >11, >11, >11 days for the 40 mg/kg dose).

In a pilot study conducted in rats, hearts from Lewis rats weretransplanted into the abdominal cavity of ACI rats. Rats were dosed withISIS 11061 at 20 mg/kg for 7 days via Alzet pump. The mean allograftsurvival time for untreated rats was 8.86±0.69 days (8, 8, 9, 9, 9, 9,10 days). In rats treated with oligonucleotide, the allograft survivaltime was 15.3±1.15 days (14, 16, 16 days).

Effects of Antisense Oligonucleotide Targeted to c-raf on Smooth MuscleCell Proliferation

Smooth muscle cell proliferation is a cause of blood vessel stenosis,for example in atherosclerosis and restenosis after angioplasty.Experiments were performed to determine the effect of ISIS 11061 onproliferation of A-10 rat smooth muscle cells. Cells in culture weregrown with and without ISIS 11061 (plus lipofectin) and cellproliferation was measured 24 and 48 hours after stimulation with fetalcalf serum. ISIS 11061 (500 nM) was found to inhibit serum-stimulatedcell growth in a dose-dependent manner with a maximal inhibition of 46%and 75% at 24 hours and 48 hours, respectively. An IC50 value of 200 nMwas obtained for this compound. An unrelated control oligonucleotide hadno effect at doses up to 500 nM.

Effects of Antisense Oligonucleotides Targeted to c-raf on Restenosis inRats

A rat carotid artery injury model of angioplasty restenosis has beendeveloped and has been used to evaluate the effects on restenosis ofantisense oligonucleotides targeted to the c-myc oncogene. Bennett etal., J. Clin. Invest. 1994, 93, 820-828. This model will be used toevaluate the effects of antisense oligonucleotides targeted to ratc-raf, particularly ISIS 11061, on restenosis. Following carotid arteryinjury with a balloon catheter, oligonucleotides are administered eitherby intravenous injection, continuous intravenous administration viaAlzet pump, or direct administration to the carotid artery in a pluronicgel matrix as described by Bennett et al. After recovery, rats aresacrificed, carotid arteries are examined by microscopy and effects oftreatment on luminal cross-sections are determined.

The invention is further illustrated by the following examples which areillustrations only and are not intended to limit the present inventionto specific embodiments.

EXAMPLES Example 1 Synthesis and Characterization of Oligonucleotides

Unmodified DNA oligonucleotides were synthesized on an automated DNAsynthesizer (Applied Biosystems model 380B) using standardphosphoramidite chemistry with oxidation by iodine.β-cyanoethyldiisopropyl phosphoramidites were purchased from AppliedBiosystems (Foster City, Calif.). For phosphorothioate oligonucleotides,the standard oxidation bottle was replaced by a 0.2M solution ofH-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwisethiation of the phosphite linkages. The thiation cycle wait step wasincreased to 68 seconds and was followed by the capping step.2'-O-methyl phosphorothioate oligonucleotides were synthesized using2'-O-methyl β-cyanoethyldiisopropyl-phosphoramidites (Chemgenes, NeedhamMass.) and the standard cycle for unmodified oligonucleotides, exceptthe wait step after pulse delivery of tetrazole and base was increasedto 360 seconds. The 3'-base used to start the synthesis was a2'-deoxyribonucleotide. 2'-O-propyl oligonucleotides were prepared by aslight modification of this procedure.

2'-fluoro phosphorothioate oligonucleotides were synthesized using5'-dimethoxytrityl-3'-phosphoramidites and prepared as disclosed in U.S.patent application Ser. No. 463,358, filed Jan. 11, 1990, and Ser. No.566,977, filed Aug. 13, 1990, which are assigned to the same assignee asthe instant application and which are incorporated by reference herein.The 2'-fluoro oligonucleotides were prepared using phosphoramiditechemistry and a slight modification of the standard DNA synthesisprotocol: deprotection was effected using methanolic ammonia at roomtemperature.

After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides were purified by precipitation twiceout of 0.5M NaCl with 2.5 volumes ethanol. Analytical gelelectrophoresis was accomplished in 20% acrylamide, 8M urea, 45 mMTris-borate buffer, pH 7.0. Oligodeoxynucleotides and theirphosphorothioate analogs were judged from electrophoresis to be greaterthan 80% full length material.

Example 2 Northern Blot Analysis of Inhibition of c-raf mRNA Expression

The human urinary bladder cancer cell line T24 was obtained from theAmerican Type Culture Collection (Rockville Md.). Cells were grown inMcCoy's 5A medium with L-glutamine (Gibco BRL, Gaithersburg Md.),supplemented with 10% heat-inactivated fetal calf serum and 50 U/ml eachof penicillin and streptomycin. Cells were seeded on 100 mm plates. Whenthey reached 70% confluency, they were treated with oligonucleotide.Plates were washed with 10 ml prewarmed PBS and 5 ml of Opti-MEMreduced-serum medium containing 2.5 μl DOTMA. Oligonucleotide withlipofectin was then added to the desired concentration. After 4 hours oftreatment, the medium was replaced with McCoy's medium. Cells wereharvested 24 to 72 hours after oligonucleotide treatment and RNA wasisolated using a standard CsCl purification method. Kingston, R. E., inCurrent Protocols in Molecular Biology, (F. M. Ausubel, R. Brent, R. E.Kingston, D. D. Moore, J. A. Smith, J. G. Seidman and K. Strahl, eds.),John Wiley and Sons, N.Y. Total RNA was isolated by centrifugation ofcell lysates over a CsCl cushion. RNA samples were electrophoresedthrough 1.2% agarose-formaldehyde gels and transferred to hybridizationmembranes by capillary diffusion over a 12-14 hour period. The RNA wascross-linked to the membrane by exposure to UV light in a Stratalinker(Stratagene, La Jolla, Calif.) and hybridized to random-primed ³²P-labeled c-raf cDNA probe (obtained from ATCC) or G3PDH probe as acontrol. RNA was quantitated using a Phosphorimager (Molecular Dynamics,Sunnyvale, Calif.).

Example 3 Specific Inhibition of c-raf Kinase Protein Expression in T24Cells

T24 cells were treated with oligonucleotide (200 nM) and lipofectin atT=0 and T=24 hours. Protein extracts were prepared at T=48 hours,electrophoresed on acrylamide gels and analyzed by Western blot usingpolyclonal antibodies against c-raf (UBI, Lake Placid, N.Y.) or A-raf(Transduction Laboratories, Knoxville, Tenn.). Radiolabeled secondaryantibodies were used and raf protein was quantitated using aPhosphorimager (Molecular Dynamics, Sunnyvale Calif.).

Example 4 Antisense Inhibition of Cell Proliferation

T24cells were treated on day 0 for two hours with various concentrationsof oligonucleotide and lipofectin (50 nM oligonucleotide in the presenceof 2 μg/ml lipofectin; 100 nM oligonucleotide and 2 μg/ml lipofectin;250 nM oligonucleotide and 6 μg/ml lipofectin or 500 nM oligonucleotideand 10 μg/ml lipofectin). On day 1, cells were treated for a second timeat desired oligonucleotide concentration for two hours. On day 2, cellswere counted.

Example 5 Effect of ISIS 5132 on T24 Human Bladder Carcinoma TumorXenografts in Nude Mice

5×10⁶ T24 cells were implanted subcutaneously in the right inner thighof nude mice. Oligonucleotides (ISIS 5132 and an unrelated controlphosphorothioate oligonucleotide suspended in saline) were administeredthree times weekly beginning on day 4 after tumor cell inoculation. Asaline-only control was also given. Oligonucleotides were given byintraperitoneal injection. Oligonucleotide dosage was 25 mg/kg. Tumorsize was measured and tumor volume was calculated on the eleventh,fifteenth and eighteenth treatment days.

Example 6 Effect of ISIS 5132 on MDA-MB 231 Human Breast Carcinoma TumorXenografts in Nude Mice

5×10⁶ MDA-MB 231 cells were implanted subcutaneously in the right innerthigh of nude mice. Oligonucleotides (ISIS 5132 and an unrelated controlphosphorothioate oligonucleotide suspended in saline) were administeredonce daily beginning on day 10 after tumor cell inoculation. Asaline-only control was also given. Oligonucleotides were given byintravenous injection at a dosage of 0.6 mg/kg or 6.0 mg/kg. Tumor sizewas measured and tumor volume was calculated on days 10, 13, 16, 20, 23and 27 following tumor cell inoculation.

For intraperitoneal oligonucleotide administration, oligonucleotideswere administered once daily beginning on day 10 after tumor cellinoculation. A saline-only control was also given. Oligonucleotides weregiven by intraperitoneal injection at a dosage of 0.6 mg/kg or 6.0mg/kg. Tumor size was measured and tumor volume was calculated on days10, 13, 16, 20, 23 and 27 following tumor cell inoculation.

Example 7 Effect of ISIS 5132 on Colo 205 Human Colon Carcinoma TumorXenografts in Nude Mice

5×10⁶ Colo 205 cells were implanted subcutaneously in the right innerthigh of nude mice. Oligonucleotides (ISIS 5132 and an unrelated controlphosphorothioate oligonucleotide suspended in saline) were administeredonce per day beginning on day 5 after tumor cell inoculation. Asaline-only control was also given. Oligonucleotides were given byintravenous injection. Oligonucleotide dosage was 6 mg/kg. Tumor sizewas measured and tumor volume was calculated on days 5, 8, 11, 14, 18,22 and 25 after tumor inoculation.

Example 8 Diagnostic Assay for raf-associated Tumors Using Xenografts inNude Mice

Tumors arising from raf expression are diagnosed and distinguished fromother tumors using this assay. A biopsy sample of the tumor is treated,e.g., with collagenase or trypsin or other standard methods, todissociate the tumor mass. 5×10⁶ tumor cells are implantedsubcutaneously in the inner thighs of two or more nude mice. Antisenseoligonucleotide (e.g., ISIS 5132) suspended in saline is administered toone or more mice by intraperitoneal injection three times weeklybeginning on day 4 after tumor cell inoculation. Saline only is given toa control mouse. Oligonucleotide dosage is 25 mg/kg. Tumor size ismeasured and tumor volume is calculated on the eleventh treatment day.Tumor volume of the oligonucleotide-treated mice is compared to that ofthe control mouse. The volume of raf-associated tumors in the treatedmice are measurably smaller than tumors in the control mouse. Tumorsarising from causes other than raf expression are not expected torespond to the oligonucleotides targeted to raf and, therefore, thetumor volumes of oligonucleotide- treated and control mice areequivalent.

Example 9 Detection of Raf Expression

Oligonucleotides are radiolabeled after synthesis by ³² p labeling atthe 5' end with polynucleotide kinase. Sambrook et al., MolecularCloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989,Volume 2, pg. 11.31-11.32. Radiolabeled oligonucleotides are contactedwith tissue or cell samples suspected of raf expression, such as tumorbiopsy samples or skin samples where psoriasis is suspected, underconditions in which specific hybridization can occur, and the sample iswashed to remove unbound oligonucleotide. Radioactivity remaining in thesample indicates bound oligonucleotide and is quantitated using ascintillation counter or other routine means.

Radiolabeled oligonucleotides of the invention are also used inautoradiography. Tissue sections are treated with radiolabeledoligonucleotide and washed as described above, then exposed tophotographic emulsion according to standard autoradiography procedures.The emulsion, when developed, yields an image of silver grains over theregions expressing raf. The extent of raf expression is determined byquantitation of the silver grains.

Analogous assays for fluorescent detection of raf expression useoligonucleotides of the invention which are labeled with fluorescein orother fluorescent tags. Labeled DNA oligonucleotides are synthesized onan automated DNA synthesizer (Applied Biosystems model 380B) usingstandard phosphoramidite chemistry with oxidation by iodine.β-cyanoethyldiisopropyl phosphoramidites are purchased from AppliedBiosystems (Foster City, Calif.). Fluorescein-labeled amidires arepurchased from Glen Research (Sterling Va.). Incubation ofoligonucleotide and biological sample is carried out as described forradiolabeled oligonucleotides except that instead of a scintillationcounter, a fluorimeter or fluorescence microscope is used to detect thefluorescence which indicates raf expression.

Example 10 A549 Xenografts

5×10⁶ A549 cells were implanted subcutaneously in the inner thigh ofnude mice. Oligonucleotides (ISIS 5132 and a scrambled raf controlphosphorothioate oligonucleotide, ISIS 10353) suspended in saline wereadministered once daily by intravenous injection at doses ranging from0.006 to 6.0 mg/kg. Resulting tumors were measured on days 9, 12, 17 and21 and tumor volumes were calculated.

Example 11 Effect of Oligonucleotide on Endogenous c-raf Expression

Mice were treated by intraperitoneal injection at an oligonucleotidedose of 50 mg/kg on days 1, 2 and 3. On day 4 animals were sacrificedand organs removed for c-raf mRNA assay by Northern blot analysis. Fourgroups of animals were employed: 1) no oligonucleotide treatment(saline); 2) negative control oligonucleotide ISIS 1082 (targeted toherpes simplex virus; 3) negative control oligonucleotide 4189 (targetedto mouse protein kinase C-a; 4) ISIS 11061 targeted to rodent c-raf.

Example 12 Cardiac Allograft Rejection Model

Hearts were transplanted into the abdominal cavity of rats or mice (of adifferent strain from the donor) as primary vascularized graftsessentially as described by Isobe et al., Circulation 1991, 84,1246-1255. Oligonucleotides were administered by continuous intravenousadministration via a 7-day Alzet pump. Cardiac allograft survival wasmonitored by listening for the presence of a second heartbeat in theabdominal cavity.

Example 13 Proliferation Assay Using Rat A-10 Smooth Muscle Cells

A10 cells were plated into 96-well plates in Dulbecco's modified Eaglemedium (DMEM)+10% fetal calf serum and allowed to attach for 24 hours.Cells were made quiescent by the addition of DMEM+0.2% dialyzed fetalcalf serum for an additional 24 hours. During the last 4 hours ofquiescence, cells were treated with ISIS 11061+lipofectin (Gibco-BRL,Bethesda Md.) in serum-free medium. Medium was then removed, replacedwith fresh medium and the cells were stimulated with 10% fetal calfserum. The plates were the placed into the incubator and cell growth wasevaluated by MTS conversion to formozan (Promega cell proliferation kit)at 24 and 48 hours after serum stimulation. A control oligonucleotide,ISIS 1082 (an unrelated oligonucleotide targeted to herpes simplexvirus), was also tested.

Example 14 Rat Carotid Artery Restenosis Model

This model has been described by Bennett et al., J. Clin. Invest. 1994,93, 820-828. Intimal hyperplasia is induced by balloon catheterdilatation of the carotid artery of the rat. Rats are anesthetized andcommon carotid artery injury is induced by passage of a balloonembolectomy catheter distended with 20 ml of saline. Oligonucleotidesare applied to the adventitial surface of the arterial wall in apluronic gel solution. Oligonucleotides are dissolved in a 0.25%pluronic gel solution at 4° C. (F127, BASF Corp.) at the desired dose.100 μl of the gel solution is applied to the distal third of the commoncarotid artery immediately after injury. Control rats are treatedsimilarly with gel containing control oligonucleotide or nooligonucleotide. The neck wounds are closed and the animals allowed torecover. 14 days later, rats are sacrificed, exsanguinated and thecarotid arteries fixed in situ by perfusion with paraformaldehyde andglutaraldehyde, excised and processed for microscopy. Cross-sections ofthe arteries are calculated.

In an alternative to the pluronic gel administration procedure, rats aretreated by intravenous injection or continuous intravenous infusion (viaAlzet pump) of oligonucleotide.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (iv) ANTI-SENSE: Yes                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      TCCCGCCTGTGACATGCATT20                                                        __________________________________________________________________________

What is claimed is:
 1. A method of inhibiting the expression of humanraf comprising contacting human tissues or cells which express human rafwith an oligonucleotide consisting of SEQ ID NO:1.
 2. A method ofinhibiting hyperproliferation of human cells comprising contacting humanhyperproliferating cells with an oligonucleotide consisting of SEQ IDNO:1.
 3. A method of treating an abnormal proliferative conditioncomprising contacting a human, or cells, tissues or a bodily fluid ofsaid human, suspected of having an abnormal proliferative condition witholigonucleotide consisting of SEQ ID NO:1.